Molecular mechanisms underlying the ferroptosis-induced epileptiform activity in mouse cortical slices

Sara Petrillo^1,2Federica Loia^1,2Michela Giustizieri³Caterina Torda^1,2Sara Cairoli^2,4Bianca Goffredo^2,4Marina Trivisano⁵Luca De Palma⁵Federico Vigevano⁶Nicola Specchio⁵Enrico Cherubini³Fiorella Piemonte^1,2*

• ¹Unit of Muscular and Neurodegenerative Diseases, Rome, Italy
• ²Bambino Gesù Children's Hospital (IRCCS), Rome, Italy
• ³Fondazione EBRI Rita Levi-Montalcini, Rome, Italy
• ⁴Research Unit of Metabolic Diseases, Rome, Italy
• ⁵Neurology, Epilepsy and Movement Disorders Unit, Bambino Gesù Children’s Hospital, IRCCS, Full Member of European Reference Network on Rare and Complex Epilepsies EpiCARE, Rome, Italy, Rome, Italy
• ⁶Developmental Disabilities Department, IRCCS San Raffaele, Rome, Italy., Rome, Italy

Ferroptosis, a newly defined iron-dependent programmed cell death, characterized by excessive accumulation of lipid peroxides and reactive oxygen species, is involved in epilepsy, particularly in those forms resistant to drugs. In a previous study, we have demonstrated that exposure of mouse cortical slices to the the ferroptosis inducer RSL-3, induces interictal epileptiform discharges. Therefore, to investigate the mechanisms underlying ferroptosis-induced epileptic activity in RSL-3-treated cortical slices, we analysed the expression of the main contributors to ferroptosis susceptibility in cells. In cortical neurons, the ferroptosis induction by RSL-3 was associated with a reduced expression of the GPX4/GSH redox pathway, responsible for the clearance of lipid peroxides, and an upregulation of 15-LOX, which promotes the formation of lipid peroxides. Moreover, in cortical slices, the cysteine/glutamate antiporter Xc-, a modulator of excitotoxicity in the brain, was up-regulated either after RSL-3-treatment or by incubating neurons with 4-HNE, the bioactive product of lipid peroxidation. Interestingly, 4-HNE was able to generate spontaneous interictal bursts, indicating a direct link between lipid peroxidation and Xc-activity, and supporting the antiporter as a potential target for treating ferroptosis-mediated drug-resistant forms of epilepsy.